DESCRIPTION: (provided by the applicant): Our studies employ as a model system the African trypanosomes, the agents of sleeping sickness in man and nagana in cattle. In the last years, trypanosomatid protozoa have attained the status of model systems for unicellular pathogens, as underscored with the advent of genome sequencing projects. Furthermore, the survey of RNA metabolism in these organisms has been instrumental in the discovery of new concepts in eukaryotic RNA biology, such as polycistronic transcription, trans-splicing of pre-mRNAs, and mitochondrial RNA editing. The major goals of this proposal remain to gain a detailed understanding of the mechanism and biological significance of RNA capping in Trypanosoma brucei. The rationale guiding our studies is as follows. First, in T. brucei m7G addition to the 5' end of RNA molecules is quite distinct from that of other eukaryotic organisms. Capping occurs on RNA polymerase (pol) II transcripts, namely the spliced leader (SL) RNA, as well as a specific subset of pol III transcripts, namely U1, U2, U3 and U4 snRNAs, but not on other more abundant pol III transcripts, raising fundamental questions about the specificity of transcript selection. Second, the presence of two distinct guanylyltransferase polypeptides in trypanosomes, each linked to a different domain, represents a novel physical organization of the capping apparatus and points to unique feature(s) of RNA capping in these organisms. Third, the identification of the nuclear cap binding complex raises questions about how selection of transcripts for nuclear export takes place in T. brucei, as both SL RNA and mature nuclear mRNA possess the same cap. For the next funding period the proposed studies aim at: 1. Understanding structural-functional aspects of the components of the capping machinery in order to define their biological role in RNA metabolism. 2. Defining sequence determinants in various capped RNAs in order to decipher the molecular details underlying the specificity of the capping reaction. 3. Beginning an analysis of the interaction between the m7G cap and the nuclear cap binding complex in order to link the cap structure to downstream events, including trans-splicing and export of capped mRNA from the nucleus into the cytoplasm.